Methods and apparatus for identification of disease associated mutations

ABSTRACT

In some embodiments, a non-transitory processor-readable medium includes code to cause a processor to receive a set of variants identified by a comparison of a test DNA sequence with a reference DNA sequence and associate at least one of the set of variants with at least one of a set of annotations each indicative of at least one criterion. The code includes code to cause the processor to filter, based on the set of annotations, the set of variants to identify a subset of variants from the set of variants. Each variant from the subset of variants is associated with at least one common annotation from the set of annotations. The code further includes code to cause the processor to present the subset of variants such that the subset of variants can be used to render a clinical diagnosis.

BACKGROUND

While the costs associated with genomic sequencing have droppeddramatically over the last several years, the costs and labor associatedwith data analysis have remained relatively constant. Thus, there is agreat need for tools to support clinical genetics and enable theanalysis of an individual's genomic data, for example, in identifyinggenetic variations potentially associated with a disease phenotype.

SUMMARY

In some embodiments, a non-transitory processor-readable medium includescode to cause a processor to receive a set of genetic variantsidentified by a comparison of an experimental sample DNA sequence with areference DNA sequence and associate at least one of the set of variantswith at least one of a set of functional or genomic annotations eachindicative of at least one criterion. The code includes code to causethe processor to filter, based on the set of annotations, the set ofvariants to identify a subset of variants from the set of variants. Eachvariant from the subset of variants is associated with at least onecommon annotation from the set of annotations. The code further includescode to cause the processor to present the subset of variants such thatthe subset of variants can be used to render a clinical diagnosis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart illustrating a sequencing analysis method,according to an embodiment.

FIG. 2 is a schematic diagram that illustrates communication devices incommunication with a host device via a network, according to anembodiment.

FIG. 3 is a schematic illustration of a processor of a host device,according to an embodiment.

FIG. 4 is a detailed view of an annotation module and a database used inannotating variants, according to an embodiment.

FIG. 5 is an illustration of a user interface associated with filteringvariants, according to an embodiment.

FIG. 6 is an illustration of a user interface associated with a detailedview of a gene and variant, according to an embodiment.

FIGS. 7A and 7B illustrate a user interface that displays a detailedview of cross sample variant filtering, according to an embodiment.

FIG. 8 is a flow chart illustrating a tertiary analysis phase method,according to an embodiment.

DETAILED DESCRIPTION

The invention provides methods for analysis of DNA sequence data andassociated software and computer systems. The method, which is generallycomputer implemented, enables a clinical geneticist or other healthcaretechnician to sift through vast amounts of DNA sequence data, toidentify potential disease-causing genomic variants. In some cases, theDNA sequence data is from a patient who may be suspected of having agenetic disorder.

Therefore, in one aspect, the invention provides a method foridentifying a genetic disorder in an individual, or identifying agenetic variant that is causative of a phenotype in an individual. Themethod comprises determining a DNA sequence for a patient suspected ofhaving a genetic disorder, identifying sequence variants, annotating theidentified variants based on one or more criteria, and filtering orsearching the variants at least partially based on the annotations, tothereby identify potential disease-causing variants.

In some embodiments, the sequence is obtained by use of a sequencinginstrument, or alternatively, DNA sequence data is obtained from anothersource, such as for example, a commercial sequencing service provider.The term “DNA sequence” as used herein refers not only to chromosomalsequence, but also to cDNA sequence, or any nucleotide sequenceinformation that allows for detection of genetic disease. Generally, theamount of sequence information is such that computational tools arerequired for data analysis. For example, the sequence data may representat least half of the individual's genomic or cDNA sequence (e.g., of arepresentative cell population or tissue), or the individuals entiregenomic or cDNA sequence. In various embodiments, the sequence datacomprises the nucleotide sequence for at least 1 million base pairs, atleast 10 million base pairs, or at least 50 million base pairs. Incertain embodiments, the DNA sequence is the individual's exome sequenceor full exonic sequence component (i.e., the exome; sequence for each ofthe exons in each of the known genes in the entire genome). Further, thesource of genomic DNA or cDNA may be any suitable source, and may be asample particularly indicative of a disease or phenotype of interest,including blood cells (e.g, PBMCs, or a T-cell or B-cell population). Incertain embodiments, the source of the sample is a tissue or sample thatis potentially malignant.

As used herein, “whole genome sequence” includes the entire sequence(including all chromosomes) of an individual's germline genome. In someembodiments, the concatenated length for a whole genome sequence isapproximately 3.2 Gbases or 3.2 billion nucleotides.

The DNA sequence may be determined by any suitable method. For example,the DNA sequence may be a cDNA sequence determined by clonalamplification (e.g., emulsion PCR) and sequencing. Base calling may beconducted based on any available method, including Sanger sequencing(chain termination), pH sequencing, pyrosequencing,sequencing-by-hybridization, sequencing-by-ligation, etc. The sequencingoutput data may be subject to quality controls, including filtering forquality (e.g., confidence) of base reads. Exemplary sequencing systemsinclude 454 pyrosequencing (454 Life Sciences), Illumina (Solexa)sequencing, SOLiD (Applied Biosystems), and Ion Torrent Systems' pHsequencing system.

The DNA sequence is mapped with one or more reference sequences toidentify sequence variants. For example, the base reads are mappedagainst a reference sequence, which in various embodiments is presumedto be a “normal” non-disease sequence. The DNS sequence derived from theHuman Genome Project is generally used as a “premier” referencesequence. A number of mapping applications are known, and includeGSMAPPER, ELAND, MOSAIK, and MAQ. Various other alignment tools areknown, and could also be implemented to map the base reads.

Based on the sequence alignments, and mapping results, sequence variantsare identified. Types of variants include insertions, deletions, indels(a colocalized insertion and deletion), translocations, inversions, andsubstitutions. While the type of variants analyzed are not limited, themost numerous of the variant types will be single nucleotidesubstitutions, for which a wealth of data is currently available. Invarious embodiments, comparison of the test sequence with the referencesequence will produce at least 500 variants, at least 1000 variants, atleast 3,000 variants, at least 5,000 variants, at least 10,000 variants,but in some embodiments, will produce at least 1 million variants, atleast three million variants, or at least 10 million variants. The toolsprovided herein enable the user to navigate the vast amounts of geneticdata to identify potentially disease-causing variants.

A wealth of data is extracted for the identified variants, including oneor more of conservation scores, genic/genomic location, zygosity, SNPID, Polyphen and SIFT predictions, splice site predictions, amino acidproperties, disease associations, annotations for known variants,variant or allele frequency data, and gene annotations. Data may becalculated and/or extracted from one or more internal or externaldatabases. Since certain categories of annotations (e.g., amino acidproperties/PolyPhen and SIFT data) are dependent on a nature of theregion of the genome in which they are contained (e.g., whether avariant is contained within a region translated to give rise to an aminoacid sequence in a resultant protein), these annotations can be carriedout for each known transcript. Exemplary external databases include OMIM(Online Mendelian Inheritance in Man), HGMD (The Human Gene MutationDatabse), PubMed, PolyPhen, SIFT, SpliceSite, reference genomedatabases, the University of California Santa Cruz (UCSC) genomedatabase, the BioBase biological databases, the dbSNP Short GeneticVariations database, the Rat Genome Database (RGD), and/or the like.Various other databases may be employed for extracting data onidentified variants. Variant information may be further stored in acentral data repository, and the data extracted for future sequenceanalyses.

Based on extracted information, the variants are annotated to facilitatefiltering of the variants, such that the variants meeting certaincriteria can be easily identified. In some embodiments, variants areinterpreted as being benign, pathogenic, or variants of unknownsignificance, based upon available information including for example,information from human mutational databases, and data stored in acentral repository. In some embodiments, the variants are annotated asmeeting, for example, one or more of the following criteria:

-   -   (i) the variant is reported to cause a disorder and recognized        to cause a disorder,    -   (ii) the variant is unreported but would be expected to cause a        disorder,    -   (iii) the variant is unreported and of the type that might be        causative of a disorder,    -   (iv) the variant is unreported and unlikely to cause a disorder,    -   (v) the variant is reported and is a recognized neutral variant,        and/or    -   (vi) the variant is unknown or not expected to be causative of        disease, but is associated with clinical presentation.

Alternative or additional bases for annotation may be employed, as longas the annotations essentially categorize variants as having a knownassociation with disease, having a known neutral effect, or having anunknown effect. For variants having an unknown effect, varioussub-categories are usually preferred to identify or score variants basedon a predicted biological impact. In various embodiments, theannotations are assigned to sequence variants on the basis of a changein encoded amino acid, a change in an RNA processing site, orinformation from a human gene mutational database. For example, thevariants may lead to a change in the encoded amino acid at one or morepositions (a non-conservative substitution), and thus the annotation maytake into account the damage prediction as a result of such amino acidchange to determine whether a resulting phenotype change (and aresulting disorder) is likely. In some embodiments, annotations can beproduced for all known transcripts. Annotations may be conductedautomatically, as described in detail below, or conducted by independentexamination, or both.

Once variants have been annotated, the variants can be filtered, basedon the annotations, in response to user queries to identify potentialdisease causing variants, and/or to confirm or rule out the presence ofa genetic disorder. Alternatively, one can browse variants according toother criteria of interest, including browsing or searching for variantsin one or more genes of interest or a genetic locus of interest, orbrowsing or searching for variants with predicted biological impact,which may be based on polypeptide damage predictions, splice sitepredictions, known gene-disease associations, or matching with variantsstored in a central data repository with patient phenotype information,or variant frequency. As described in detail herein, the invention incertain aspects provides a user interface to enable such filtering andsearching.

Variants may be tagged by the user with additional descriptiveinformation to aid subsequent analysis. For example, confidence in theexistence of the variant can be recorded as confirmed, preliminary, orsequence artifact. Certain sequencing technologies have a tendency toproduce certain types of sequence artifacts, and the invention allowssuch suspected artifacts to be recorded. The variants may be furthertagged in basic categories of benign, pathogenic, or unknown, or aspotentially of interest.

The method may employ a computer-readable medium, or “non-transitoryprocessor-readable medium.”

In particular, queries can be run to identify variants meeting certaincriteria, or variant report pages can be browsed by chromosomal positionor by gene, the latter allowing researchers to focus on only thosevariations that exist in a particular set of genes of interest. In someembodiments, the user selects only variants with well-documented andpublished disease associations (e.g., by filtering based on HGMD orother disease annotation). Alternatively, the user can filter forvariants not previously associated with disease, but of a type likely tobe deleterious, such as those introducing frameshifts, non-synonymoussubstitutions (predicted by Polyphen or SIFT), or prematureterminations. Further, the user can exclude from analysis those variantsbelieved to be neutral (based on their frequency of occurrence instudies populations), for example, through exclusion of variants indbSNP. Additional exclusion criteria include mode of inheritance (e.g.,heterozygosity), depth of coverage, and quality score.

In certain embodiments, base calling is carried out to extract thesequence of the sequencing reads from an image file produced by aninstrument scanner. Following base calling and base qualitytrimming/filtering, the reads are mapped against a reference sequence(assumed to be normal for the phenotype under analysis) to identifyvariations (variants) between the two with the assumption that one ormore of these differences will be associated with phenotype of theindividual whose DNA is under analysis. Subsequently, each variant isannotated with data that can be used to determine the likelihood thatthat particular variant is associated with the phenotype under analysis.The analysis may be fully or partially automated as described in detailbelow, and may include use of a central repository for data storage andanalysis, and to present the data to analysts and clinical geneticistsin a format that makes identification of variants with a high likelihoodof being associated with the phenotypic difference more efficient andeffective.

In some embodiments, a user is been provided with the ability to runcross sample queries where the variants from multiple samples areinterrogated simultaneously. In such embodiments, for example, a usercan build a query to return data on only those variants that are exactlyshared across a user defined group of samples. This can be useful forfamily based analyses where the same variant is believed to beassociated with disease in each of the affected family members. Foranother example, the user can also build a query to return only thosevariants that are present in genes where the gene contains at least one,but not necessarily the same, variant. This can be useful where a groupof individuals with disease are not related (the variants associatedwith the disease are not necessary exactly the same, but result in acommon alteration in normal function). For yet another example, the usercan specify to ignore genes containing variants in a user defined groupof samples. This can be useful to exclude polymorphisms (variantsbelieved or confirmed not to be associated with disease) where the userhas access to a user defined group of control individuals who arebelieved to not have the disease associated variant. For each of thesequeries a user can additionally filter the variants by specifying any orall of the previously discussed filters (using the advanced searchshown, for example, in FIG. 5) on top of the cross sample analyses. Thisallows a user to identify variants matching these criteria, which areshared between or segregated amongst samples.

FIG. 1 is a flow chart illustrating a sequencing analysis method 10. Thesequencing analysis method 10 includes a primary analysis phase 40, asecondary analysis phase 50 and a tertiary analysis phase 60. As shownin FIG. 1, the primary analysis phase 40 includes deriving a test DNAsequence associated with an individual, at 12. In some embodiments, forexample, an instrument scanner can produce an image file associated withthe individual's DNA sequence. Base calling can then be performed toextract the test DNA sequence from the image file. Base quality trimmingand filtering can then be performed on the test DNA sequence, at 14.

The secondary analysis phase 50 includes mapping the test DNA sequenceagainst a reference DNA sequence, at 16, and identifying variationsand/or differences (variants) between the test DNA sequence and thereference DNA sequence, at 18. In some embodiments, the reference DNAsequence can be deemed “normal” and/or “a baseline” for one or morephenotypes, disorders and/or diseases under analysis. Thus, anassumption can be made that one or more variants between the test DNAsequence and the reference DNA sequence can be associated with thephenotype, disorder and/or disease under analysis. In some embodiments,the test DNA sequence and/or the reference DNA sequence is an entirehuman genome. In other embodiments, the test DNA sequence and/or thereference DNA sequence includes between 1 million base pairs and 50million base pairs. In still other embodiments, the test DNA sequenceand/or the reference DNA sequence includes fewer than 1 million basepairs or greater than 50 million base pairs.

In some embodiments, any suitable application, tool and/or system can beused to perform the primary analysis phase 40 and/or the secondaryanalysis phase 50. In some embodiments, for example, a tool such asRoche's GSMAPPER, ELAND, SourceForge.net's MAQ (Mapping and Assemblingwith Qualities), Boston College's MOSAIK and/or the like can be used toperform the primary analysis phase 40 and/or the secondary analysisphase 50. These tools can output a result that includes the identifiedvariants that can be input into a variant analysis system thatimplements the tertiary analysis phase 60. Different tools can outputthe results of the secondary analysis phase 50 in different file formatsand/or syntaxes. As such, the variant analysis system can be configuredto receive various file formats and/or syntaxes of variant data andconvert the various file formats and/or syntaxes of variant data into acommon file format or syntax prior to performing the tertiary analysisphase 60.

The tertiary analysis phase 60 includes annotating the variants, at 20.Specifically, each variant can be annotated with data that can be usedto determine a likelihood that that particular variant is associatedwith the phenotype, disorder and/or disease under analysis. A variantcan be annotated, for example, with functional information, amino acidproperties, conservation scores, zygosity data, allele frequencies,quality scores, disease associations, PolyPhen damage predictions, achange in an RNA processing site, splice site predictions, or otherinformation from a human gene mutational database. Additionally, in someembodiments, each variant can be annotated with data that indicateswhether (1) the variant has been reported to cause a disorder andrecognized to cause a disorder; (2) the variant is unreported butexpected to cause a disorder; (3) the variant is unreported and of thetype that might be causative of a disorder; (4) the variant isunreported and unlikely to cause a disorder; (5) the variant has beenreported and is a recognized neutral variant; and/or (6) the variant isunknown or not expected to be causative of disease but has a knownassociation with clinical presentation. Such annotations are extractedfrom existing datasets and/or calculated using the variant analysissystem.

The variants can then be filtered based on these annotations, at 22 andthe filtered variants can be presented to a user (e.g., a clinicalgeneticist) for use in a clinical diagnosis, at 24. For example, a usercan filter the variants using the annotations such that variants meetinga specific criteria are presented. The user can then use these variantsin connection with rendering a clinical diagnosis.

FIG. 2 is a schematic diagram that illustrates communication devices 180in communication with a host device 120 via a network 170 to implement avariant analysis system, according to an embodiment. Specifically,communication device 150 is configured to communicate with the hostdevice 120. Similarly, communication device 160 is configured tocommunicate with the host device 120. The network 170 can be any type ofnetwork (e.g., a local area network (LAN), a wide area network (WAN), avirtual network, a telecommunications network, the Internet) implementedas a wired network and/or wireless network. As described in furtherdetail herein, in some embodiments, for example, the communicationdevices 180 are personal computers connected to the host device 120 viaan internet service provider (ISP) and the Internet (e.g., network 170).

The host device 120 can be any type of device configured to send dataover the network 170 to and/or receive data from one or more of thecommunication devices 180. In some embodiments, the host device 120 canbe configured to function as, for example, a server device (e.g., a webserver device), a network management device, and/or so forth.

The host device 120 includes a memory 124 and a processor 122. Thememory 124 can be, for example, a random access memory (RAM), a memorybuffer, a hard drive, and/or so forth. In some embodiments, the memory124 of the host device 120 includes data used to facilitate a variantanalysis system. In such embodiments, for example, the host device 120is configured to execute code that implements the variant analysissystem. As such, the host device 120 can send data associated with thevariant analysis system to and receive data associated with the variantanalysis system from the communication devices 180. For example, asdescribed in further detail herein, the host device 120 can send dataassociated with a result of a variant analysis to the communicationdevices 180. Additionally, the host device 120 can receive dataassociated with a variant filtering request from a communication device180. In some embodiments, the host device 120 can receive data from thecommunication devices 180 pertaining to variant analysis. For example,the host device can receive an indication that a user of a communicationdevice 180 wishes to import variant data, a user of a communicationdevice 180 wishes to annotate variant data, a user of a communicationdevice 180 wishes to filter variant data, and/or the like.

In some embodiments, the memory 124 of the host device 120 storesaccount information associated with users of the variant analysissystem. In such embodiments, for example, the host device 120 can storea username and password associated with a user, preferences associatedwith the user, a listing of variant analyses conducted by the user,and/or the like. In other embodiments, such information is stored in adatabase (e.g., database 126) operatively coupled to the host device120.

The database 126 is operatively coupled to the host device 120. In someembodiments, the data associated with the variant analysis (e.g.,variant annotation data, variant identification data, data associatedwith diseases and/or phenotypes, specific variant data imported to thememory 124 by a user of a communication device 180, and/or the like) isstored in the database 126. In such embodiments, the host device 120 canquery the database 126 for data associated with the variant analysis.Specifically, when a user wishes to view data associated with a variantanalysis, a communication device 180 can send a request for data to thehost device 120; the host device 120 can query the database 126 for therequested data; and the host device 120 can send the requested data tothe communication device 180.

In some embodiments, for example, the database 126 can be an Oracle 11 gdatabase and/or the like. In some embodiments, the host device 120 canfacilitate communication between the communication devices and thedatabase 126 using a middleware layer. In some embodiments, such amiddleware layer can be a Java middleware layer and the host device 120can be a Java application server.

Each of the communication devices 180 can be, for example, a computingentity (e.g., a personal computing device such as a desktop computer, alaptop computer, etc.), a mobile phone, a personal digital assistant(PDA), and/or so forth. Although not shown, in some embodiments, each ofthe communication devices 180 can include one or more network interfacedevices (e.g., a network interface card) configured to connect thecommunication devices 180 to the network 170. In some embodiments, thecommunication devices 180 can be referred to as client devices.

As shown in FIG. 2, the communication device 160 has a processor 162, amemory 164, and a display 166. The memory 164 can be, for example, arandom access memory (RAM), a memory buffer, a hard drive, and/or soforth. The display 166 can be any suitable display, such as, forexample, a liquid crystal display (LCD), a cathode ray tube display(CRT) or the like. Similar to communication device 160, thecommunication device 150 has a processor 152, a memory 154, and adisplay 156.

In some embodiments, a web browser application can be stored in thememory 164 of the communication device 160. Using the web browserapplication, the communication device 160 can send data to and receivedata from the host device 120. Similarly, the communication device 150can include a web browser application. In such embodiments, thecommunication devices 180 act as thin clients. This allows minimal datato be stored on the communication devices 180. In other embodiments, thecommunication devices 180 can include an application specific tocommunicating with the host device 120 when using the variant analysissystem. In such embodiments, the communication devices 180 can downloadthe application from the host device 120 prior to running the variantanalysis system. In some embodiments, such an application can be, forexample, an Adobe Flex application executing on the host device 120using a web browser application.

As discussed above, the communication devices 180 can send data to andreceive data from the host device 120 associated with a variant analysissystem. In some embodiments, the data sent between the communicationdevices 180 and the host device 120 can be formatted using any suitableformat. In some embodiments, for example, the data can be formattedusing extensible markup language (XML), hypertext markup language (HTML)and/or the like.

In some embodiments, one or more portions of the host device 120 and/orone or more portions of the communication devices 180 can include ahardware-based module (e.g., a digital signal processor (DSP), a fieldprogrammable gate array (FPGA)) and/or a software-based module (e.g., amodule of computer code to be executed at a processor, a set ofprocessor-readable instructions that can be executed at a processor). Insome embodiments, one or more of the functions associated with the hostdevice 120 (e.g., the functions associated with the processor 122) canbe included in one or more modules (see, e.g., FIG. 3). In someembodiments, one or more of the functions associated with thecommunication devices 180 (e.g., functions associated with processor 152or processor 162) can be included in one or more modules. In someembodiments, one or more of the communication devices 180 can beconfigured to perform one or more functions associated with the hostdevice 120, and vice versa.

FIG. 3 is a schematic illustration of a processor 200 of a host device(e.g., host device 120 of FIG. 2), according to another embodiment. Theprocessor 200 includes a data input module 202, an annotation module204, a filter module 206, a presentation module 208, a communicationmodule 210 and a user annotation module 212. Such modules can beconfigured to implement a variant analysis system for performing atertiary analysis phase (e.g., tertiary analysis phase 60 of FIG. 1) ofa sequencing analysis method (e.g., sequencing analysis method 10 ofFIG. 1).

Such modules can be hardware-based modules (e.g., a digital signalprocessor (DSP), a field programmable gate array (FPGA)) and/orsoftware-based modules (e.g., a module of computer code to be executedat processor 200, a set of processor-readable instructions that can beexecuted at a processor 200). While each module is shown in FIG. 3 asbeing in direct communication with every other module, in otherembodiments, each module need not be in direct communication with everyother module. For example, the data input module 202 might not be indirect communication with the presentation module 210.

The communication module 210 can facilitate communication between theprocessor 200 of the host device and one or more communication devices(e.g., communication devices 180 of FIG. 2). Accordingly, the othermodules of the processor 200 can use the communication module 210 tosend data to and receive data from the communication devices.Additionally, in some embodiments, the communication module 210 canfacilitate communication between the processor 200 of the host deviceand one or more databases (e.g., database 126 of FIG. 2). Accordingly,the processor 200 can send data, queries, and/or the like to a databasevia communication module 210. Similarly, the processor 200 can receivedata in response to queries via the communication module 210.

The data input module 202 is configured to receive data from acommunication device (e.g., via communication module 210). In someembodiments, such data can include variant data associated with aspecific individual, variant annotation data, and/or the like. Variantdata can include variations identified by mapping a test DNA sequenceagainst a reference DNA sequence as produced in a secondary analysisphase of a sequencing analysis method, as described above with respectto FIG. 1. In some embodiments, the test DNA sequence and/or thereference DNA sequence is an entire human genome. In other embodiments,the test DNA sequence and/or the reference DNA sequence includes between1 million base pairs and 50 million base pairs.

Variant annotation data can include information and/or identifiedrelationships used to annotate variants. In some embodiments, the datainput module 202 can send the data to a database (e.g., database 126)for storage and future retrieval and/or use. In other embodiments, theinput module 202 can send the data to another module (e.g., annotationmodule 204) for further processing and/or analysis.

As discussed above, in some embodiments, different secondary analysisphase tools output variant data in different file formats and/or syntax.Accordingly, in some embodiments, the data input module 202 can beconfigured to receive variant data in multiple file formats and/orsyntaxes. The data input module 202 can then convert the variant datainto a single common format and/or syntax. For example, the data inputmodule 202 can be configured to convert variant data received in a firstfile format and/or syntax and produced by a first secondary analysisphase tool to a second file format and/or syntax. Similarly, the datainput module 202 can be configured to convert variant data received in athird file format and/or syntax and produced by a second secondaryanalysis phase tool to the same second file format and/or syntax. Theremaining portions of the variant analysis system can be configured toread and/or analyze the variant data in the common format and/or syntax.

The annotation module 204 is configured to receive variant data from thedata input module 202 and associate the variants associated with thevariant data with annotations. For example, each variant can beannotated with data that can be used to determine a likelihood that thatparticular variant is associated a disease, disorder and/or phenotypeunder analysis. The annotation module 204 can annotate a variant with,for example, functional information, amino acid properties, conservationscores, zygosity data, allele frequencies, quality scores, diseaseassociations, PolyPhen damage predictions, a change in an RNA processingsite, splice site predictions, and/or the like. Additionally, in someembodiments, the annotation module 204 can annotate one or more variantswith data that indicates whether (1) the variant has been reported tocause a disorder and recognized to cause a disorder; (2) the variant isunreported but expected to cause a disorder; (3) the variant isunreported and of the type that might be causative of a disorder; (4)the variant is unreported and unlikely to cause a disorder; (5) thevariant has been reported and is a recognized neutral variant; and/or(6) the variant is unknown or not expected to causative of disease buthas a known association with clinical presentation. In some embodiments,variants can be associated with annotations on the basis of a change inencoded amino acid, a change in an RNA processing site or informationfrom a human gene mutational database. Such annotations are extractedfrom existing datasets and/or calculated using the variant analysissystem.

FIG. 4 is a detailed view of an annotation module 220 (similar toannotation module 204 of FIG. 3) and a database 226 (e.g., similar todatabase 126) for use in annotating variants. In some embodiments, thedatabase 226 stores information used by the annotation module 220 toannotate variants. In such embodiments, the information can be stored indatabase 226 and retrieved from database 226 by the annotation moduleduring an annotation process. Specifically, the host device 220 canreceive system data 280, variant data 292 and/or sample data 290 fromthe database 226 during an annotation process. Variant data 292 can beassociated with the variants identified in a secondary phase analysisand provided to the database 226 by a communication device (e.g., viadata input module 202). Sample data 290 can be reference sequence datareceived from, for example, Illumina, 454 Life Sciences and/or the like.System data 280 can be extracted from one or more internal or externaldatabases such as, for example, an amino acid properties database, OMIM(Online Mendelian Inheritance in Man), HGMD (The Human Gene MutationDatabse), PubMed, PolyPhen, SIFT, SpliceSite, reference genomedatabases, the University of California Santa Cruz (UCSC) genomedatabase, the BioBase biological databases, the dbSNP Short GeneticVariations database, the Rat Genome Database (RGD), and/or the like.Such system data can be used to annotate the variant data 292.

The annotation module 220 includes functionality to calculate variantconsequence 282, RNA splice site predictions 284, depth of coverage 286and variant zygosity 288. In some embodiments, RNA spice site prediction284 can be calculated using GeneSplicer, Splice Site and/or any othertool or algorithm. Variant consequence 282 can be calculated usingPolyPhen, SIFT, and/or the like.

Returning to FIG. 3, after annotating the variant data, the annotationmodule 204 can store the variant data and its associated annotations ina database (e.g., database 126 of FIG. 1). This allows a user (e.g., aclinical geneticist) to perform subsequent analysis using the variantdata and the annotations.

The filter module 206 is configured to allow users (e.g., clinicalgeneticists) to filter, search and/or query the variant data forvariants having one or more commonalities. Specifically, the filtermodule 206 can be configured to receive search parameters from acommunication device (e.g., via communication module 210). For example,a user might send an instruction to the filter module 206 to search forall variants that are highly conserved, homozygous and novel (i.e., notidentified in a reference data set). For another example, a user mightsend an instruction to the filter module 206 to search for all variantsalready associated with a disease. Based on the search parameters, thefilter module 206 can provide a query to the database (e.g., database126 of FIG. 1) storing the annotated variant data. The database canidentify and return the variants matching the search parameters.

The presentation module 208 is configured to provide a graphical userinterface to a user of the variant analysis system. In some embodiments,for example, the presentation module 208 sends instructions to acommunications device (e.g., via communications module 210) to rendervarious user interfaces on a display of the communications device. Forexample, the presentation module 208 can send instructions to cause thecommunications device to provide a data input user interface, whichallows a user to load variant data, on a display of the communicationsdevice. For another example, the presentation module can sendinstructions to cause the communications device to provide a variantfilter user interface (e.g., FIG. 4) on a display of the communicationsdevice. For yet another example, the presentation module can sendinstructions to cause the communications device to provide informationassociated with a specific gene and/or variant (e.g., FIG. 5). A user ofthe variant analysis system can control and/or use the variant analysissystem via the various user interfaces of the variant analysis system.

FIG. 5, for example, is an illustration of a user interface 300associated with filtering variants based on annotations of thosevariants. User interface 300 can be provided to a communications deviceby the presentation module 208. The user can then provide search and/orfilter parameters and/or criteria to the filter module 206 using userinterface 300.

User interface 300 includes a gene search portion 310, an advancedsearch portion 320 and a search results summary portion 330. The genesearch portion 310 of user interface 300 allows a user to search forand/or filter variants by gene identifier and/or gene set identifier.For example, as shown in FIG. 5, a search can be conducted forGJA4,EXO1,ISG15,CCDC27.

The advanced search portion 320 of user interface 300 allows a user tosearch for and/or filter variants using a variety of parameters such as,for example, conservation, sift prediction, frequency, polyphenprediction, hetrozygosity, splice site, and/or the like. The advancedsearch portion 320 of user interface 300 also allows a user to searchfor and/or filter variants that are synonymous, non-synonymous, novel,known-biobase, known-dbSNP, intergenic, genic, protein coding, intronic,3 prime UTR and/or 5 prime UTR. The search results summary portion 330includes a summary of the genes identified as variants using theparameters identified in the gene search portion 310 and the advancedsearch portion 320.

FIG. 6 is an illustration of a user interface 400 that displays adetailed view of a gene and variant identified by filtering the variantdata. Similar to user interface 300, user interface 400 can be providedto a communications device by the presentation module 208. The user canthen analyze in detail the genes and variants identified by filteringthe variant data.

Specifically, user interface 400 includes a gene summary portion 410 anda variant summary portion 420. The gene summary portion 410 providesinformation associated with a particular gene such as, for example, adescription, protein identifiers, position information, and/or the like.The variant summary portion 420 includes information associated with thevariants identified for that gene. Additionally, the variant summaryportion includes a variant summary for each known transcript 430.

Returning to FIG. 3, the user annotation module 212 can receiveannotations made by users of the variant analysis system. Specifically,a user of the variant analysis system can identify the variants as beingconfirmed, preliminary and/or an artifact. Additionally, a user of thevariant analysis system can flag variants as likely to be benign,pathogenic or of unknown significance. Such user annotations can beadded to the database (e.g., database 126) such that variants can beautomatically annotated with the user provided annotations (e.g., byannotation module 204) during subsequent analysis. This allows users ofthe system to add their professional analysis to the annotated variants,leading to a clinical diagnosis.

FIGS. 7A and 7B illustrate a user interface 500 that displays a detailedview of cross sample variant filtering. Similar to user interface 300(shown and described with respect to FIG. 5), user interface 500 can beprovided to a communications device by the presentation module 208 (FIG.3). A user of the communication device can then analyze in detail thegenes and/or variants identified by filtering the variant data usingsample selection.

Specifically, user interface 500 includes a selection portion 510, amethodology portion 520, an additional filter portion 530 and a resultsportion 540. The selection portion 510 allows a user to select samplesto be analyzed 510. The methodology portion 520 allows a user of thecommunication device to select a methodology to be used for cross sampleanalysis. The additional filter portion 530 can be used to furtherdefine the methodology selected in the methodology portion 520. Thereturned cross sample variant containing genes can be presented to theuser as a table in the results portion 540. In some embodiments,additional columns 550 of data, based on the variant annotations, can beadded to the table in the results portion 540. A user can select anorder in which the data appears in the table of the results portion 540(e.g., an order of the columns) using selection inputs 560.

FIG. 8 is a flow chart illustrating a tertiary analysis phase method600, according to an embodiment. In some embodiments, the tertiaryanalysis phase method 600 can be performed by a variant analysis system.The tertiary analysis phase method 600 includes receiving a set ofvariants identified by a comparison of a test DNA sequence with areference DNA sequence, at 602. As discussed above, in some embodiments,the set of variants can be received in multiple different file formatsand/or syntaxes. In such embodiments, the set of variants can beconverted into a common file format and/or syntax that can be used inthe remaining steps of method 600.

At least one of the set of variants is associated with at least one of aset of annotations each indicative of at least one criterion, at 604. Asdiscussed above, in some embodiments, each variant can be annotated withdata that can be used to determine a likelihood that that particularvariant is associated with a disease, disorder and/or phenotype underanalysis. Additionally, such annotations can be extracted from existingdatasets and/or calculated using a variant analysis system.

The set of variants is filtered, based on the set of annotations, toidentify a subset of variants from the set of variants, at 606. Eachvariant from the subset of variants is associated with at least onecommon annotation from the set of annotations. Such filtering can bebased on any suitable criteria, such as, for example, the criteriadescribed with respect to filter module 206 of FIG. 3. In someembodiments, a user (e.g., clinical geneticist or the like) provides thecriteria to a variant analysis system performing method 600. In suchembodiments, the user can search for and/or filter variants and/or genesassociated with particular diseases and/or conditions. The subset ofvariants is then presented such that the subset of variants can be usedto render a clinical diagnosis, at 608.

While various embodiments have been described above, it should beunderstood that they have been presented by way of example only, and notlimitation. Where methods described above indicate certain eventsoccurring in certain order, the ordering of certain events may bemodified. Additionally, certain of the events may be performedconcurrently in a parallel process when possible, as well as performedsequentially as described above.

For example, while shown and described above (e.g., with respect to FIG.2) as being implemented using a host device and a network, in otherembodiments, a variant analysis system can be implemented locally. Forexample, the variant analysis system can be software stored in memory ofa personal computing device (PC) and implemented by a processor of thePC. In such embodiments, for example, the PC can download the softwarefrom a host device and/or install the software using any suitable devicesuch as a compact disc (CD).

Some embodiments described herein relate to a computer storage productwith a non-transitory computer-readable medium (also can be referred toas a non-transitory processor-readable medium) having instructions orcomputer code thereon for performing various computer-implementedoperations. The computer-readable medium (or processor-readable medium)is non-transitory in the sense that it does not include transitorypropagating signals per se (e.g., a propagating electromagnetic wavecarrying information on a transmission medium such as space or a cable).The media and computer code (also can be referred to as code) may bethose designed and constructed for the specific purpose or purposes.Examples of non-transitory computer-readable media include, but are notlimited to: magnetic storage media such as hard disks, floppy disks, andmagnetic tape; optical storage media such as Compact Disc/Digital VideoDiscs (CD/DVDs), Compact Disc-Read Only Memories (CD-ROMs), andholographic devices; magneto-optical storage media such as opticaldisks; carrier wave signal processing modules; and hardware devices thatare specially configured to store and execute program code, such asApplication-Specific Integrated Circuits (ASICs), Programmable LogicDevices (PLDs), Read-Only Memory (ROM) and Random-Access Memory (RAM)devices.

Examples of computer code include, but are not limited to, micro-code ormicro-instructions, machine instructions, such as produced by acompiler, code used to produce a web service, and files containinghigher-level instructions that are executed by a computer using aninterpreter. For example, embodiments may be implemented using Java,C++, or other programming languages (e.g., object-oriented programminglanguages) and development tools. Additional examples of computer codeinclude, but are not limited to, control signals, encrypted code, andcompressed code.

While various embodiments have been described above, it should beunderstood that they have been presented by way of example only, notlimitation, and various changes in form and details may be made. Anyportion of the apparatus and/or methods described herein may be combinedin any combination, except mutually exclusive combinations. Theembodiments described herein can include various combinations and/orsub-combinations of the functions, components and/or features of thedifferent embodiments described.

What is claimed is:
 1. A non-transitory processor-readable mediumstoring code representing instructions to be executed by a processor,the code comprising code to cause the processor to: receive a pluralityof variants identified by a comparison of a test DNA sequence with areference DNA sequence; associate at least one variant from theplurality of variants with at least one annotation from a plurality ofannotations including at least one of: (i) reported to cause a disorderand recognized to cause a disorder, (ii) unreported but expected tocause a disorder, (iii) unreported and of the type that might becausative of a disorder, (iv) unreported and unlikely to cause adisorder, (v) reported and recognized neutral variant, or (vi) unknownor not expected to be causative of disease, but associated with clinicalpresentation; filter, based on the plurality of annotations, theplurality of variants to identify a set of variants from the pluralityof variants, each variant from the set of variants being associated withat least one common annotation from the plurality of annotations; andpresent the set of variants such that the set of variants can be used torender a clinical diagnosis.
 2. The non-transitory processor-readablemedium of claim 1, wherein the code includes code to cause the processorto filter for variants annotated as: having been reported to cause adisorder and recognized to cause a disorder.
 3. The non-transitoryprocessor-readable medium of claim 1, wherein the code includes code tocause the processor to filter for variants annotated as: unreported butexpected to cause a disorder.
 4. The non-transitory processor-readablemedium of claim 1, wherein the code includes code to cause the processorto filter for variants annotated as: unreported and of the type thatmight be causative of a disorder.
 5. The non-transitoryprocessor-readable medium of claim 1, wherein the code includes code tocause the processor to filter for variants annotated as: unreported andunlikely to cause a disorder.
 6. The non-transitory processor-readablemedium of claim 1, wherein the code includes code to cause the processorto filter for variants annotated as: reported and recognized neutralvariant.
 7. The non-transitory processor-readable medium of claim 1,wherein the code includes code to cause the processor to filter forvariants annotated as: unknown or not expected to be causative ofdisease, but having a known association with clinical presentation. 8.The non-transitory processor-readable medium of any one of claims 2 to7, wherein variants are associated with annotations on the basis of achange in encoded amino acid, a change in an RNA processing site, orinformation from a human gene mutational database.
 9. The non-transitoryprocessor-readable medium of claim 8, wherein the variants involving achange in encoded amino acid include predictions of phenotype change.10. The non-transitory processor-readable medium of claim 1, wherein theat least one variant is a single nucleotide polymorphism.
 11. Thenon-transitory processor-readable medium of claim 1, wherein the atleast one variant is a rare or infrequent allele.
 12. The non-transitoryprocessor-readable medium of claim 1, wherein the at least one variantis identified by genomic location, zygosity, allele frequency and/or SNPID.
 13. The non-transitory processor-readable medium of claim 1, whereinthe code to cause the processor to receive includes code to cause theprocessor to receive the plurality of variants identified by thecomparison of the test DNA sequence of at least 1 million base pairswith the reference DNA sequence of at least 1 million base pairs. 14.The non-transitory processor-readable medium of claim 1, wherein thecode to cause the processor to receive includes code to cause theprocessor to receive the plurality of variants identified by thecomparison of the test DNA sequence of at least 10 million base pairswith the reference DNA sequence of at least 10 million base pairs. 15.The non-transitory processor-readable medium of claim 1, wherein thecode to cause the processor to receive includes code to cause theprocessor to receive the plurality of variants identified by thecomparison of the test DNA sequence of at least 50 million base pairswith the reference DNA sequence of at least 50 million base pairs. 16.The non-transitory processor-readable medium of claim 1, wherein thecode to cause the processor to receive includes code to cause theprocessor to receive the plurality of variants identified by thecomparison of the test DNA sequence of an entire human genome with thereference DNA sequence of the entire human genome.
 17. Thenon-transitory processor-readable medium of claim 1, wherein the code tocause the processor to receive includes code to cause the processor toreceive the plurality of variants identified by the comparison of thetest DNA sequence of a human exome with the reference DNA sequence of ahuman exome.
 18. The non-transitory processor-readable medium of claim1, wherein the test DNA sequence is a cDNA sequence, and/or isdetermined by clonal amplification of cDNA of an individual suspected ofhaving a genetic disorder.
 19. The non-transitory processor-readablemedium of claim 1, wherein the code further comprises code to cause theprocessor to tag variants as: confirmed, preliminary, or sequenceartifact.
 20. The non-transitory processor-readable medium of claim 1,wherein the plurality of variants includes at least 1 million variants.21. The non-transitory processor-readable medium of claim 1, wherein theplurality of variants includes at least 10 million variants.
 22. Thenon-transitory processor-readable medium of claim 1, wherein theplurality of variants includes at least 15 million variants.
 23. Thenon-transitory processor-readable medium of claim 1, wherein the codefurther comprises code to cause the processor to flag variants asbenign, pathogenic, or unknown.
 24. The non-transitoryprocessor-readable medium of claim 23, wherein the code furthercomprises code to cause the processor to filter variants tagged asbenign, pathogenic, or unknown.
 25. The non-transitoryprocessor-readable medium of claim 1, wherein the code includes code tocause the processor to filter variants in response to user inputs. 26.The non-transitory processor-readable medium of claim 19 or 23, whereinthe code includes code to cause the processor to tag or flag variants inresponse to user inputs.
 27. The non-transitory processor-readablemedium of claim 1, wherein the plurality of variants is a firstplurality of variants, the code to cause the processor to receive thefirst plurality of variants includes code to cause the processor toreceive the first plurality of variants in a first syntax, the code tocause the processor to: receive, in a second syntax different from thefirst syntax, a second plurality of variants identified by a comparisonof a third nucleotide sequence with the second nucleotide sequence;convert the first plurality of variants to a third syntax; and convertthe second plurality of variants to the third syntax.
 28. Thenon-transitory processor-readable medium of claim 1, wherein the code tocause the processor to associate includes code to cause the processor toassociate the at least one variant from the plurality of variants withthe at least one annotation from the plurality of annotations using aplurality of external databases.
 29. A method for identifying a geneticdisorder in an individual, comprising: determining a DNA sequence for apatient suspected of having a genetic disorder, comparing the DNAsequence with one or more reference sequences to identify a plurality ofvariants, annotating, at an annotation module implemented in at leastone of a memory or a processing device, each variant from the pluralityof variants with at least one annotation from a set of annotationsincluding at least one of: (i) reported to cause a disorder andrecognized to cause a disorder, (ii) unreported but expected to cause adisorder, (iii) unreported and of the type that might be causative of adisorder, (iv) unreported and unlikely to cause a disorder, (v) reportedand recognized neutral variant, or (vi) unknown or not expected to becausative of disease, but associated with clinical presentation,filtering the plurality of variants on the basis of the set ofannotations; and identifying the presence or absence of the geneticdisorder.
 30. The method of claim 29, wherein annotations from the setof annotations are assigned on the basis of a change in encoded aminoacid, a change in an RNA processing site, or information from a humangene mutational database.
 31. The method of claim 29, wherein theplurality of variants involving a change in encoded amino acid includepredictions of phenotype change.
 32. The method of claim 29, wherein atleast one variant from the plurality of variants is a single nucleotidepolymorphism.
 33. The method of claim 29, wherein at least one variantfrom the plurality of variants is a rare or infrequent allele.
 34. Themethod of claim 29, wherein the plurality of variants are identified bygenomic location, zygosity, allele frequency and/or SNP ID.
 35. Themethod of claim 29, wherein at least half of the individual's DNAsequence is determined.
 36. The method of claim 29, wherein the DNAsequence of at least 1 million base pairs is determined.
 37. The methodof claim 29, wherein the DNA sequence of at least 10 million base pairsis determined.
 38. The method of claim 29, wherein the DNA sequence ofat least 50 million base pairs is determined.
 39. The method of claim29, wherein an exome sequence is determined.
 40. The method of claim 29,wherein the DNA sequence is a cDNA sequence, and/or is determined byclonal amplification of cDNA.
 41. The method of claim 29 furthercomprising, tagging the plurality of variants as confirmed, preliminary,or sequence artifact.
 42. The method of claim 29, wherein the pluralityof variants includes at least three million variants.
 43. The method ofclaim 29, wherein the plurality of variants includes at least 10 millionvariants.
 44. The method of claim 29, wherein the plurality of variantsincludes at least 15 million variants.
 45. The method of claim 29,further comprising, flagging variants from the plurality of variants asbenign, pathogenic, or unknown.
 46. The method of claim 29, implementedusing a non-transitory processor-readable medium.